ISSN 1392-2130. VETERINARIJA IR ZOOTECHNIKA (Vet Med Zoot). T. 44 (66). 2008
DIVERSITY OF THERMOPHILIC Campylobacter ISOLATED FROM SLAUGHTER PIGS IN NORWAY Olav Rosef1, Algimantas Paulauskas2, Aud Stølan1, Elisabeth Moen Bråthen1, Camilla Haslekås1 1 Telemark University College, Telemark, Norway, 2 Vytautas Magnus University, Kaunas, Lithuania Correspondence: Olav Rosef, Telemark University College, Hallvard Eikas Plass 1, 3800 Bø i Telemark, Norway Fax: +47 35952703. E-mail:
[email protected], Telephone +47 35952782 Summary. Out of 100 faecal swab samples, 88 thermophilic Campylobacter representing ten herds were isolated. The species were identified by biotyping and multiplex polymerase chain reaction (PCR). Of the isolates, 86 (97.7%) were C. coli, one was C. lari, and one was C. jejuni. All three species were isolated from the same herd. Automatic riboprints were performed with the PstI restriction enzyme and RiboPrinter® (automated microbial characterization system). The identification of the isolates was predicted when the corresponding pattern matched one of the patterns in the DuPont identification (DUP-ID) library and was then assigned an identification number. A total of 81 selected C. coli isolates were riboprinted. The isolates could be divided into two clades and five subclades, and 19 isolates could be given five different DUP-IDs from the library: DUP-PST1-1182 (n=6), DUP-PST1-1208 (n=10), DUP-PST1-1175 (n=1), DUP-PST1-1140 (n=1), and DUP-PST1-1163 (n=1). Pigs are healthy carriers of C. coli which showed a high degree of diversity among and within the herds. Keywords: Campylobacter, diversity, pig, clustering, genotyping, riboprinting.
TERMOFILINIŲ Campylobacter IZOLIATŲ IŠ KIAULIŲ SKERDIENOS ĮVAIROVĖ NORVEGIJOJE Olav Rosef1, Algimantas Paulauskas2, Aud Stølan1, Elisabeth Moen Bråthen1, Camilla Haslekås1 1 Telemark University College, Hallvard Eikas Plass 1, 3800 Bø i Telemark, Norway; faks.: +47 3595 2703; tel. +47 3595 2782; el. paštas:
[email protected] 2 Vytauto Didžiojo universitetas, Vileikos g. 8, Kaunas, Lietuva Santrauka. Iš 100 kiaulių fekalijų tepinėlių 88 rastos termofilinės Campylobacter. Rūšis nustatyta biotipuojant ir dauginės PGR metodais: 86 (97,7 proc.) pavyzdžiuose nustatyta C. coli, po vieną C. lari ir C. jejuni. Visos trys bakterijos rūšys išskirtos iš tos pačios bandos gyvulių. Automatinis genotipavimas atliktas naudojant restrikcijos fermentą PstI ir prietaisą „RiboPrinter®“. Tirtos bakterijos įvertintos lyginant su DuPont identifikavimo (DUP-ID) bibliotekos katalogo pavyzdžiais, joms suteiktas atitinkamas identifikacijos numeris. „RiboPrinter®“ prietaisu genotipavus 81 C. coli pavyzdį, atlikta klasterinė analizė parodė, kad tirti pavyzdžiai pasiskirsto į dvi sankaupas (klasterius) su penkiais subklasteriais. 19 pavyzdžių priklauso penkiems skirtingiems žinomiems DUP-ID bibliotekos katalogo genotipams: DUPPST1-1182 (n=6), DUP-PST1-1208 (n=10), DUP-PST1-1175 (n=1), DUP-PST1-1140 (n=1) ir DUP-PST1-1163 (n=1). Kiaulių pernešama C. Coli pasižymi didele įvairove tiek tarp skirtingų bandų, tiek ir bandos viduje. Raktažodžiai: Campylobacter, įvairovė, genotipavimas, prietaisas „RiboPrinter®“, kiaulės. apparently fragile organisms that are unable to grow in the presence of air or multiply outside the animal host and are highly susceptible to a number of environmental conditions (Park 2002). Murphy et al. (2006) summarize the mechanisms by which Campylobacter spp. adapt to stress conditions and thereby increase their ability to survive on food and in the environment. Reliable and powerful typing methods for campylobacters are necessary in order to gain more insight into infection routes. Traditionally, phenotyping methods such as serotyping and biotyping have been used. The drawbacks of these methods are their restricted resolutions, the lack of specific reagents for serotyping, and a large portion of untypable strains. Using genotyping to determine genetic relatedness is necessary to provide data for a better understanding of the epidemiological aspects. The purpose of this study was to identify the thermophilic campylobacters isolated from pigs to species level and to use the automated PstI ribotyping method (restriction enzyme polymorphism analysis of automated microbial characterization system) to provide evidence of genetic
Introduction. Thermophilic campylobacters, represented by Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis, are zoonotic with many animals serving as reservoirs for human diseases including rabbits, rodents, wild birds, sheep, horses, cows, pigs, poultry, and domestic pets (Rosef et al.1983, Kapperud and Rosef 1983, Brown et al. 2004, Johnsen et al. 2006a). The incidence of human cases of Campylobacter infection has increased and recently exceeded that of Salmonella in many European countries (http://www.efsa.europa.eu/en.html) and is the most frequently reported cause of bacterial gastrointestinal illness. In industrialized countries, cases of human campylobacteriosis due to C. jejuni and C. coli are about 90% and 10% respectively. Despite the high number of human cases, knowledge of the pathogenesis and epidemiology of infections is still incomplete. Pigs are natural reservoirs of campylobacters, and isolation rates have been found to be between 77.6% and 100%, dominated by C. coli with 90.1 to 100% (Rosef 1981ab, Rosef et al.1983, Guevremont et al. 2004, Boes et al. 2005). Campylobacter are 62
ISSN 1392-2130. VETERINARIJA IR ZOOTECHNIKA (Vet Med Zoot). T. 44 (66). 2008 determined when the corresponding patterns matched one of the patterns of the DuPont Identification library of the RiboPrinter® with a similarity of > 0.85. The PstI ribotype patterns were automatically assigned a DuPont identification number (e.g. DUP-PST1-1182) by the RiboPrinter®, which was confirmed by visual inspection. The profiles were transferred and analyzed with the GelComparII® software (Bio Numerics, Applied Maths inc.) using the Pearson correlation and default settings for optimalization (2.0%) and position tolerance (1.00%) for genetic similarity. The dendrogram was generated by the unweighted pair group method with arithmetic averages (UPGMA) to determine profile relatedness. Results and discussion Out of 100 faecal swab samples, 88 thermophilic Campylobacter (88%), representing all the herds investigated, were isolated (Table 1). Both biochemical and multiplex PCR distinguished the isolates to species level (Table 2). Out of 88 isolates, 86 (97.7%) were C. coli, one was C. lari, and one was C. jejuni as shown in Table 2. Both C .jejuni and C. lari were isolated from the same herd. (Table 1). Pigs are healthy carriers of C. coli and is also isolated from pigs environment and as surface contaminant of pig carcasses (Rosef , 1981ab) The low isolation rate of thermophilic campylobacters others than C. coli is in accordance with earlier findings (Rosef et al. 1983, Guévremont et al 2004, Boes et al. 2005).
relatedness. We used a highly standardized method coupled with a computer-based pattern analysis in order to compare the strains, which then provided the possibility to compare ribotype profiles and the strains in a dendrogram. Material and methods Bacterial isolates Faecal swabs (Invasive sterile Eurotube® Collection Swab) from pigs (n=100) representing 10 herds were collected at slaughter and streaked onto modified bloodfree CCDA-Preston agar (Oxoid CM 739, Oxoid SR 155, and Oxoid SR 2342 E). The plates were incubated at 42ºC under microaerobic conditions using Oxoid CampyGen in an anaerobic jar. The plates were read after 24 and 48 hours and typical colonies confirmed by phase contrast microscopy. The isolates (n=88) were kept frozen (-70ºC) in Microbank™ (Pro-Lab Diagnostics, Canada) until further examination. Biochemical differentiation The isolates were subcultivated and controlled for purity by phase contrast microscopy and by growth on nonselective blood agar plates. To distinguish among the species, the method described by Hwang and Ederer (1975) for detecting hydrolysis of hippurate was used. Susceptibility to nalidixic acid was evaluated on blood agar plates using antibiotic disks containing 30 µg nalidixic acid (Oxoid Limited, Hampshire, England). PCR-assay The multiplex-PCR primer sets described by Wang et al. (2002) were used for species confirmation of the isolates. The colony multiplex-PCR was optimized to simultaneously identify the 23S rRNA from C. spp., the hipO gene from C. jejuni, and the glyA gene from C. coli, C. lari, and C. upsaliensis. In short, a loopful of colony material from a single colony was suspended in 500µL of water, centrifuged, and 5 µL of the supernatant used as a template in the polymerase chain reaction (PCR). The PCR-mix consisted of the species-specific primers and multiplex master mix (Qiagen GmbH, Hilden, Germany), water, and the template in a total volume of 50µL. The PCR was run (Bio-Rad, iCycler) with the following conditions: 95ºC for 6 min, 30 repetitions of 95ºC for 30 sec, 56ºC for 30sec, 72ºC for 30 sec, 72ºC for 7 min, and finally ending at 4ºC. The PCR products were run on a 1.5 % agarose gel with TAE and ethidium bromide and visualized under UV light. C. coli, C. jejuni, C. lari, and C. upsaliensis strains were used as positive controls. Riboprinting Ribotyping was performed on 81 selected C. coli isolates using the DuPont Qualicon RiboPrinter® as previously described (Bruce 1996). Single colonies from a 24h culture on blood agar plates were suspended in sample buffer and heated at 80ºC for 15 min. After the addition of lytic enzymes, samples were transferred to the RiboPrinter®. Further analysis for the restriction of DNA, including use of the PstI enzyme (20.000U/ML, New England Biolabs), was carried out automatically. The riboprint profiles were aligned according to the position of a molecular size standard and compared with patterns stored in the library. The identification of an isolate was
Table 1. Isolation of Campylobacter species from the herds Herds A B C D* E F G H I J
Number of samples 9 1 1 39 1 1 5 1 15 27
Campylobacter Isolated 8 1 1 31 1 1 5 1 13 26
* One isolate of C. jejuni, and one C. lari was isolated from herd D. Although thermophilic campylobacters do not multiply outside their natural habitat, they may survive fairly well in the external environment, especially in aquatic niches (Blaser et al. 1980, Thomas et al. 1999). Because of the lack of the possibility for campylobacters to multiply in the environment, the epidemiological question is how long they can survive in the environment and cause infections. At present, this is unknown. The infective dose, however, is very low, with only a few hundred bacteria necessary to cause human infection (Black et al. 1988). The high infection rate indicates that hygienic precautions should be taken when handling pigs. 63
ISSN 1392-2130. VETERINARIJA IR ZOOTECHNIKA (Vet Med Zoot). T. 44 (66). 2008
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Fig 1. Riboprint diversity of 81 strains C. coli isolated from pigs at slaughter
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ISSN 1392-2130. VETERINARIJA IR ZOOTECHNIKA (Vet Med Zoot). T. 44 (66). 2008 Table 2. Distribution of Campylobacter species by using biotyping and multiplex PCR Species C. jejuni C. coli C. lari Total
Campylobacter multiplex PCR 1 86 1 88
Biotyping Hippurate Nalidixic acid 1 0 0 0 0 1 1 1
% 1,1 97,7 1,1 100
DuPont ID 19 19
Earlier works on the serotyping of C. spp. isolated among domestic and wild animals (including pigs) have displayed diversity with 42 different serotypes among the typeable strains (65,7%) (Rosef et al.1985). Knowledge of the virulence and diversity of the strains isolated from pigs is lacking. We used the restriction enzyme PstI for automatic ribotyping because of the Qualicon library identification (DUP-Pst ID) in the RiboPrinter® and because of the use by other researchers (de Boer 2000, Ge et al. 2006). It is then possible to compare the ribotype pattern with other RiboPrinter® users. A number of different ribotypes were found within C. coli. The ribotypes could be divided into two clades and five subclades (a-e) as showed in Figure 1. Five strains did not fall into clusters. Nineteen isolates could be given five different Dup-IDs from the library. DUP-PSTl-1182 (n=5), DUP-PSTl-1208 (n=10), and DUP-PSTl-1175 (n=1) - representing 16 of the DUP-IDs classified - were in subcluster b originating from five herds. Subcluster e with 15 isolates represents one herd. One of these strains was given a DUP-ID (DUP- PST1-1140). One strain in subcluster d was assigned DUP-PST1-1163 (Figure 1). It could be concluded that there is a high diversity of the C. coli isolates among and within the herds. There are, however, few common ribotypes between isolates from human cases and pigs (Rosef et al. not published). Since the factors responsible for virulence are unknown, it is quite possible that many environmental isolates may not be pathogenic - even though they belong to the same species, are serologically identical, and belong to the same ribogroup as clinical isolates. Due to the lack of detectable virulence factors and the high number of isolates from pigs, special precautions should be taken to avoid the risk of transmitting campylobacteriosis.
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ISSN 1392-2130. VETERINARIJA IR ZOOTECHNIKA (Vet Med Zoot). T. 44 (66). 2008 15. Rosef, O.. Isolation of Campylobacter fetus subsp. jejuni from the gallbladder of normal slaughter pigs, using an enrichment procedure. 1981b. Acta vet. scand. 22: 149-151. 16. Rosef, O., Gondrosen, B., Kapperud, G. & Underdal, B. Isolation and characterization of Campylobacter jejuni and Campylobacter coli from domestic and wild mammals in Norway. 1983. Appl. Environ. Microbiol. 46: 855-859. 17. Rosef, O., Kapperud, G., Lauwers, S. & Gondrosen, B. Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals. 1985. Appl. Environ. Microbiol. 49: 1507-1510. 18. Thomas, C., Hill, D.J. & Mabey, M. Evaluation of temperature and nutrients on the survival of Campylobacter spp. in water microcosms. 1999. J. Appl. Microbiol. 86: 1024-1032. 19. Wang, G., Clark, C.G., Taylor, T.M., Pucknell, C., Barton, C., Price, L., Woodward, D.L. & Rodgers, F.G. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsalensis and C. fetus.subsp. fetus. 2002. J. Clin. Microbiol. 40: 4744-4747. Received 1 July 2008 Accepted 17 October 2008
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